The V5 tag is derived from a small epitope (Pk) found on the P and V proteins of the paramyxovirus of simian virus 5 (SV5). The V5 tag is generally used with all 14 amino acids (GKPIPNPLLGLDST), but it may also be used with a shorter 9-amino acid (IPNPLLGLD) sequence.

V5 tag antibodies provide a dependable method for the detection and purification of tagged target proteins without a protein-specific antibody or probe. Invitrogen V5 tag antibodies are designed to reliably detect recombinant V5-tagged proteins, and each antibody is validated for use in many applications.

Flow cytometry analysis of V5 tag was done on HEK-293 cells transiently overexpressing V5-His-LacZ. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with V5 Tag Mouse Monoclonal Antibody (Cat. No. 377500, red histogram) or with mouse isotype control (pink histogram) at a dilution of 1:250 in 2.5% BSA. After incubation at room temperature for 3 hours, the cells were labeled with Alexa Fluor 488 Rabbit Anti-Mouse Secondary Antibody (Cat. No. A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Western blot analysis of V5. Analysis was performed by loading 25 µg of 293T cells transfected with 12Tag onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4°C overnight. The membrane was probed with a V5 epitope tag polyclonal antibody (Cat. No. PA1-993) at a dilution of 1:2,000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hour at room temperature in the dark. Chemiluminescent detection was performed using Thermo Scientific Pierce ECL Plus Western Blotting Substrate (Cat. No. 32132). Results show a band at ~45 kDa.